Project Summary/Abstract For more than three decades, research supported by this grant has had a major impact on our understanding of how Sinorhizobium meliloti invades nodules and establishes the chronic intracellular infection that underlies the symbiosis with its legume host. Our work has also identified common bacterial functions that are important for both symbiotic and pathogenic bacteria to interact with their respective eukaryotic hosts. In addition, it has also led to unanticipated fundamental discoveries, including the missing enzyme in vitamin B12 biosynthesis, and YbeY, a previously unrecognized, extremely highly conserved endoribonuclease that plays key roles in bacterial RNA metabolism and has human and plant homologs. The proposed research builds on our past progress to address critical issues, including how plant-encoded defensin-like NCR (Nodule Cysteine Rich) peptides modulate S. meliloti's cell cycle and physiology during symbiosis, and how the RNase YbeY exerts its multiple biological roles. NCR peptides play key roles in the striking process in which the bacteria undergo rounds of endoreduplication and terminally differentiate into bacteroids. We will continue to evaluate the symbiotic and antimicrobial activities of chemically synthesized NCR peptides and their variants and also representative antimicrobial peptides; continue to develop and exploit a novel strategy to observe the physiological consequences of expressing NCR peptides, antimicrobial peptides, or variants in different subcellular locations; and continue to use crosslinking to identify direct targets of NCR peptides. Our prior research has identified cellular functions that enable S. meliloti to respond appropriately to NCR peptides while resisting their antimicrobial action. We will build on our prior discovery and analysis of the symbiotically critical BacA protein and numerous other functions identified in our recent Tn-seq study. We will analyze structure- function relationships of the symbiotically important S. meliloti BacA protein; establish the membrane topology of BacA and collaborate to obtain a crystal structure; and characterize other bacterial proteins that affect the antimicrobial activity of NCR247 and assess their possible importance for the S. meliloti-legume symbiosis. We initially identified YbeY because of its critical role in symbiosis and have subsequently shown that it is a single- strand endoribonuclease that plays key roles in 16S rRNA 3'-processing, 70S ribosome quality control, and small RNA regulation. We will continue our investigations of the mechanism of action and physiological importance of YbeY. We will complete our in vitro study of the role of YbeY in the maturation of the 3' terminus of 16S rRNA; determine the nature of the ribosomal defects in a ?ybeY strain; identify global RNA processing targets of YbeY; and test whether any known inhibitors of Mycobacterium tuberculosis act by targeting YbeY.